NEUROSCIENCE
Bright and photostable chemigenetic
indicators for extended in vivo
voltage imaging
Ahmed S. Abdelfattah^1 , Takashi Kawashima^1 †, Amrita Singh1,2, Ondrej Novak1,3,
Hui Liu^1 , Yichun Shuai^1 , Yi-Chieh Huang^4 , Luke Campagnola^5 , Stephanie C. Seeman^5 ,
Jianing Yu^1 , Jihong Zheng^1 , Jonathan B. Grimm^1 , Ronak Patel^1 , Johannes Friedrich6,7,8,
Brett D. Mensh^1 , Liam Paninski6,7, John J. Macklin^1 , Gabe J. Murphy^5 ,
Kaspar Podgorski^1 , Bei-Jung Lin^4 , Tsai-Wen Chen^4 , Glenn C. Turner^1 , Zhe Liu^1 ,
Minoru Koyama^1 , Karel Svoboda^1 , Misha B. Ahrens^1 ‡,
Luke D. Lavis^1 ‡, Eric R. Schreiter^1 ‡§
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity
at high spatial and temporal resolution. However, the utility of existing GEVIs has been
limited by the brightness and photostability of fluorescent proteins and rhodopsins.
We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes
instead of protein-based fluorophores, thereby extending the number of neurons imaged
simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations
relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish,
and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and
subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute
period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of
spike timing with behavior.
A
nimal behavior is produced by patterns of
neuronal activity that span a wide range of
spatial and temporal scales. Understand-
ing how neural circuits mediate behavior
thus requires high-speed recording from
ensembles of neurons for long periods of time.
Although the activity of large numbers of neu-
rons can now be routinely recorded using ge-
netically encoded calcium indicators (GECIs) ( 1 ),
the slow kinetics of calcium signals complicate
the measurement of action potentials, and sub-
threshold voltage signals are missed entirely ( 1 – 3 ).
Voltage imaging using genetically encoded volt-
age indicators (GEVIs) can overcome these chal-
lenges, enabling imaging of fast spikes and
subthreshold dynamics in genetically defined
neurons ( 4 , 5 ). The high imaging speed and
excitation intensity required for voltage imag-
ing, combined with the smaller volume of the
cellular membrane, place increased demands on
voltage indicators relative to GECIs. Extant GEVIs
rely on fluorescence fromeither microbial rho-
dopsins ( 6 – 8 ) or fluorescent proteins ( 9 – 13 ).
These fluorophores lack the brightness and photo-
stability to allow in vivo voltage imaging from
large fields of view over time scales of many be-
havioral events, precluding the millisecond–time
scale analysis of neural circuits. Improved rho-
damine dyes such as the Janelia Fluor (JF) dyes
can be used in complex biological experiments be-
cause of their high brightness and photostability
( 14 ), compatibility with self-labeling protein tags
( 15 , 16 ), and ability to traverse the blood-brain
barrier for in vivo delivery ( 17 ). We describe a
“chemigenetic,”or hybrid protein–small mole-
cule, GEVI scaffold that we call Voltron, which
irreversibly binds these synthetic fluorophore
dyes. Voltron provides an increased photon yield
that enables in vivo imaging of neuronal spiking
and subthreshold voltage signals in model or-
ganisms with order-of-magnitude improvement
in the number of neurons imaged simultane-
ously over substantially longer durations.
Our design for a chemigenetic voltage indica-
tor combines a voltage-sensitive microbial rho-
dopsin domain ( 6 , 7 , 11 ) with a dye-capture protein
domain (Fig. 1A) that irreversibly binds a syn-
thetic fluorophore dye ligand ( 14 , 15 ) (Fig. 1B),
analogous to previously reported voltage indi-
cators that use fluorescent proteins ( 10 , 11 , 18 ).
Transmembrane voltage–dependent changes in
the absorption spectrum ( 6 , 19 ) of the rhodopsin
domain of Voltron reversibly modulate the de-
gree of fluorescence quenching of the nearby
bound dye through Förster resonance energytransfer (FRET). We investigated the modular-
ity of this approach, finding that three differ-
ent rhodopsin domains—QuasAr1 ( 7 ), QuasAr2
( 7 ), and Ace2N ( 11 , 20 )—modulated the fluores-
cence of the rhodamine dye Janelia Fluor 549
(JF 549 ) after binding to either HaloTag ( 15 )or
SNAP-tag ( 21 ) dye-capture protein domains (figs.
S1 to S8). Removing a small number of amino
acid residues at the junction of the rhodopsin
and self-labeling tag domains increased the am-
plitude of fluorescent voltage signals (fig. S1),
presumably by decreasing average distance and
thus increasing FRET efficiency between the dye
and rhodopsin retinal cofactor. The configura-
tion providing the best signal-to-noise ratio (SNR)
for spikes was Ace2N fused to HaloTag with five
amino acids removed at their junction (Fig. 1, A
and B, and fig. S2), hereafter referred to as Voltron.
We tested several Voltron-dye combinations
in cultured rat neuronsand acute mouse brain
slices with high-speed imaging and simulta-
neous whole-cell patch clamp electrophysiology
(Fig. 1C, figs. S6 and S9 to S13, and tables S1 and
S2). Voltron could detect neuronal action poten-
tials and subthreshold potential changes with a
variety of JF dye ligands with emission maxima
between 520 nm and 660 nm using fluorescence
imaging with one-photon excitation (Fig. 1, C to
E, and fig. S6) but was not compatible with two-
photon imaging, as described previously for
rhodopsin-containing GEVIs ( 22 , 23 ). Voltron
bound to JF 525 (Voltron 525 ) exhibited the highest
sensitivity, giving a fluorescence change of–23 ±
1%DF/F 0 for a voltage step from–70 mV to
+30 mV (Fig. 1E and fig. S9); Voltron 549 showed
similar sensitivity. Voltron 525 responded to volt-
age steps with submillisecond on and off time
constants (table S3 and fig. S10). We compared
the brightness and photostability of Voltron in
neuronal cultures with those of two other fluo-
rescent protein–based GEVIs: Ace2N-mNeon ( 11 )
and ASAP2f ( 13 ). Both Voltron 525 and Voltron 549
were brighter than Ace2N-mNeon (by a factor of
3to4)andASAP2f(byafactorof16to18)(Fig.
1F) in cell culture. This difference did not result
from differences in expression; we compared the
brightness of Voltron 549 and Ace2N-mNeon at
the single-molecule level and observed a similar
brightness difference (factor of 3 to 4) (Fig. 1G).
Voltron 525 and Voltron 549 were also more photo-
stable in ensemble measurements (Fig. 1H, tables
S4 and S5, and figs. S14 and S15) as well as in
single-molecule assays, in which photobleaching
times were longer for Voltron 549 than those of
Ace2N-mNeon by a factor of 8 (Fig. 1I). Overall,
the improved brightness and photostability of
Voltron increased the photon yield by at least a
factor of 10 in neurons relative to existing GEVIs
that rely on fluorescent proteins.
In vivo, Voltron could be reliably expressed
and labeled with dye in mice, larval zebrafish,
and adult fruit flies (Figs. 1 to 4 and figs. S16
to S19 and S21 to S45). Simultaneous in vivo
electrophysiology and Voltron imaging in each
of these organisms confirmed the detection of
individual action potentials (Fig. 1, J and K, and
figs.S17toS19).Forimaginginthemousebrain,RESEARCH
Abdelfattahet al.,Science 364 , 699–704 (2019) 16 August 2019 1of6
(^1) Janelia Research Campus, Howard Hughes Medical
Institute, Ashburn, VA 20147, USA.^2 Solomon H. Snyder
Department of Neuroscience, Johns Hopkins University,
Baltimore, MD 21205, USA.^3 Department of Auditory
Neuroscience, Institute of Experimental Medicine,
Academy of Sciences of the Czech Republic, Prague,
Czech Republic.^4 Institute of Neuroscience, National
Yang-Ming University, Taipei 112, Taiwan.^5 Allen Institute
for Brain Science, Seattle, WA 98109, USA.^6 Department
of Statistics and Center for Theoretical Neuroscience,
Columbia University, New York, NY 10027, USA.
(^7) Department of Neuroscience and Grossman Center for
the Statistics of Mind, Columbia University, New York, NY
10027, USA.^8 Center for Computational Biology, Flatiron
Institute, New York, NY 10010, USA.
*These authors contributed equally to this work.†Present address:
Department of Neurobiology, Weizmann Institute of Science,
Rehovot, Israel.‡These authors contributed equally to this work.
§Corresponding author. Email: [email protected]